Human complement factor I does not require cofactors for cleavage of synthetic substrates

Area of Science:

• Biochemistry
• Immunology
• Protease research

Background:

• Complement factor I (fI) is a key regulator of the complement system.
• fI circulates actively and cleaves C3b or C4b with cofactors like factor H (fH).
• No synthetic substrates for fI had been previously reported.

Purpose of the Study:

• To investigate the substrate specificity of complement factor I (fI).
• To identify novel synthetic substrates for fI activity.
• To compare fI's specificity and activity with thrombin.

Main Methods:

• Utilized peptide-7-amino-4-methylcoumarin derivatives to probe fI substrate specificity.
• Assayed fI amidolytic activity across various pH and ionic strengths.
• Tested a range of inhibitors, including synthetic thrombin inhibitors and metal ions.

Main Results:

• Identified five novel synthetic substrates cleaved by fI.
• fI exhibits specificity similar to thrombin but with significantly lower catalytic activity.
• fI's amidolytic activity has a pH optimum of 8.25, while proteolytic activity on C3b favors acidic pH (<5.5).
• fH and C3(NH3) did not affect tripeptide substrate cleavage rates.
• Specific inhibitors (thrombin inhibitor, Pefabloc SC, Suramin) and metal ions (Cr2+, Fe3+) affected fI activity.

Conclusions:

• This study establishes synthetic substrates for fI, enabling detailed specificity analysis.
• fI's distinct pH optima for amidolytic vs. proteolytic activity highlight regulatory mechanisms.
• Inhibitor studies provide insights into fI's active site and potential therapeutic targets.